Our aim is to define developmental pathways that govern the genesis of hematopoietic stem cells (HSC). The main goal of this proposal is to investigate the role of placenta in hematopoietic stem cell development. Our hypothesis is that placenta contributes to the formation, maturation and/or expansion of hematopoietic stem cells during fetal life, and thus may represent an important, previously unrecognized site for HSC development. Our temporal transplantation studies have shown a development of a major HSC niche in placenta during midgestation. 1) We will now define the immunophenotype of HSCs in placenta by transplantation of sorted cell populations from placenta and fetal hematopoietic organs into irradiated adult recipients. 2) Subsequently, we will define the spatial localization of placenta HSCs and their putative precursors in placenta sections by immunohistochemistry and confocal microscopy by using markers that will be defined by transplantation and by analyzing mouse strains where genes for critical hematopoietic transcription factors SCL or runxl/AML1 have been targeted. 3) By establishing placenta explant cultures we will address whether HSCs are generated de novo in placenta or seeded from elsewhere and investigate whether placenta microenvironment can support HSC maturation and expansion. If we can identify precursors for HSCs by functional assessment in explant cultures, we will ultimately compare these developmentally related but functionally distinct hematopoietic cell populations by expression profiling. Our goal is to identify molecular programs that are associated with the ability to reconstitute adult bone marrow and define stemness in the hematopoietic compartment. We believe that our studies will not only clarify the controversy of HSC origin during embryogenesis but also help to identify cell autonomous and microenvironmental factors supporting the genesis, maturation, and expansion of HSCs. If microenvironmental factors promoting "stemness" of HSCs during embryonic development can be harnessed to induce HSC maturation and expansion in vitro on placenta explant cultures, this culture system might potentially be used to generate or expand human HSCs for therapeutic purposes.